Comparison of the inactivation of Bacillus subtilis spores and MS2 bacteriophage by MIOX, ClorTec and hypochlorite

AIMS: To compare the disinfection ability of two widely used electrolytic generation systems (ClorTec and MIOX) and the conventional chlorine disinfectant (sodium hypochlorite) using three strains of Bacillus subtilis spores and MS2 bacteriophage. METHODS AND RESULTS: Three B. subtilis aerobic spore strains (ATCC1A1, 35021 and 35946) and the bacteriophage MS2 (ATCC 15597-B1) were propagated and sporulated. Four indicator organisms were exposed to four disinfectant treatments for comparing the effectiveness of inactivation: hypochlorite, ClorTec, MIOX and MIOX-anode. The results indicated that the two electrolytic generation systems were as effective as the conventional chlorination for the inactivation of micro-organisms used. Some data points showed the variation using anova analysis, in which the inactivation of MIOX and ClorTec was higher than that of hypochlorite. CONCLUSIONS: The ClorTec and MIOX systems are quite similar to hypochlorite in the inactivation-effectiveness for aerobic spores and bacteriophage in drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: Laboratory-scale investigation proved that gaseous chlorine could be replaced by either ClorTec or MIOX systems for the drinking water treatment utilities, which still could maintain the same disinfection efficiency.     

Genes coding for virulence determinants of Campylobacter jejuni in human clinical and cattle isolates from Alberta, Canada, and their potential role in colonization of poultry

Forty nine Campylobacter jejuni isolates from cattle feces collected from Alberta feedlots and 50 clinical C. jejuni isolates from people in Alberta were tested for the presence of 14 genes encoding putative virulence factors by PCR. These included genes implicated in adherence and colonization (flaC, cadF, docC, racR, jlpA, peb1, and dnaJ), invasion (virB11, ciaB, pldA, and iamA) and protection against harsh conditions (htrA, cbrA, and sodB). The genes examined were widely distributed in both the cattle fecal isolates and the human isolates. Of the isolates tested, 67% contained all of the genes except virB11. The cadF gene was found in 100% of the isolates tested. The presence or absence of virulence-associated genes was not associated with the ability of the organism to colonize birds. All of the C. jejuni isolates used to challenge birds were able to colonize the animals regardless of virulence gene profile. While some diversity in the profile of the occurrence of virulence-associated genes in C. jejuni exists, the distribution of these putative virulence-associated genes isolates from feedlot cattle feces and humans in Alberta was similar. In addition it was not possible to predict the ability of the selected isolates to colonize young chicks based on the presence of these genes coding for virulence determinants.     

Attenuated virulence of min operon mutants of Neisseria gonorrhoeae and their interactions with human urethral epithelial cells

Neisseria gonorrhoeae, a sexually-transmitted gram-negative bacterium, causes gonorrhoea in humans. The min genes of N. gonorrhoeae are involved in cell division site selection with oxyR co-transcribed with these genes. The mutation in min genes and oxy R cause aberrant cell morphology and aggregation patterns, respectively. Our objective was to assess the contribution of neisserial min operon cell division genes i.e. minC, minD and oxyR in virulence. Compared to the N. gonorrhoeae parental strain (Ng CH811Str(R)), its isogenic mutants with insertionally inactivated minC (Ng CSRC1), minD (Ng CJSD1) or oxyR (Ng KB1) showed reduced adherence to and invasion of urethral epithelial cells. This may be explained by defective microcolony formation in the mutant strains, possibly owing to abnormal morphology and aggregation. The expression levels of surface virulence factors like Opa, pilin and lipooligosaccharide in the mutants were unchanged relative to Ng CH811Str(R). Furthermore, in urethral epithelial cells, the min and oxyR mutants induced the release of proinflammatory cytokines like IL6 and IL8 to levels similar to that induced by the parental strain. Taken together, our studies indicate that inactivation of minC, minD or oxyR in N. gonorrhoeae attenuates its ability to bind to and invade urethral epithelial cells without altering its potential to induce IL6 and IL8 release.     

Designing nanoconjugates to effectively target pancreatic cancer cells in vitro and in vivo

Background

Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells.Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.

Methodology/Principal Findings

The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.

Conclusion

Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates.     

Fas-, caspase 8-, and caspase 3-dependent signaling regulates the activity of the aminophospholipid translocase and phosphatidylserine externalization in human erythrocytes

Apoptosis and erythrocyte senescence share the common feature of exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD), caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the raft marker proteins Galpha(s) and CD59; the existence of Fas-associated FasL, FADD and caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower aminophospholipid translocase activity and higher levels of PS externalization in comparison with young cells. In support of our contention that caspases play a functional role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events, including translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3. These events were independent of calpain but dependent on reactive oxygen species (ROS) as evident from the effects of the ROS scavenger N-acetylcysteine. Caspase activation was associated with loss of aminophospholipid translocase activity and with PS externalization. ROS was not generated by treatment of cells with t-butyl hydroperoxide at 10 degrees C, and Fas did not translocate into rafts. Concomitantly, neither formation of a Fas-associated signaling complex nor caspase activation could be observed, supporting the view that translocation of Fas into rafts was the trigger for the chain of events leading to caspase 3 activation. Our data demonstrate for the first time the novel involvement of Fas/caspase 8/caspase 3-dependent signaling in an enucleated cell leading to PS externalization, a central feature of erythrophagocytosis and erythrocyte biology.     

Oxo-transfer catalysis from t-BuOOH with C-H bond insertion using tridentate Schiff-base-chelate complexes of ruthenium(III)

Mixed-chelate complexes of ruthenium have been synthesized using tridentate Schiff-base ligands (TDLs) derived by condensation of aldehydes (salicyldehyde, 2-pyridinecarboxaldehyde) with 2-aminobenzoic acid, and bidentate ligands (2,2^′-bipyridine or picolinic acid). [Ru^III(cpsd)(bipy)(H₂O)]⁺ (1), [Ru^III(cpsd)(pic)(H₂O)] (2), [Ru^III(cppc)(bipy)(H₂O)]²⁺ (3) and [Ru^III(cppc)(pic)(H₂O)]⁺ (4) complexes (where, cpsd²⁻=(N-(2-carboxyphenyl)salicylaldiminato); cppc⁻=N-2-carboxyphenylpyridine-2-carboxaldiminato; bipy=2,2^′-bipyridine and pic⁻=picolinate) were characterized by analytical, spectral (IR and UV-Vis), conductance, magnetic moment and electrochemical studies. Catalysis of hydrocarbon oxidations for cyclohexene, cyclohexane, cyclohexanol, toluene, benzyl alcohol, and tetrahydrofuran have been studied using various O-atom transfer agents (t-BuOOH, H₂O₂, NaOCl, KHSO₅ and pyridinium-N-oxide). The influence of product yield as a function of solvent was evaluated for CH₂Cl₂, CH₃CN, and 1,4-dioxane. Coordinating solvents suppress the reactivity by inhibiting coordination of t-BuOOH, and compete for the Ru^VO group through their own intrinsic C-H reactivity. The main pathway transfers the oxo group from the [RuO(TDL)(XY)] intermediate, TDL=cpsd²⁻ and cppc²⁻; XY=bipy or pic⁻, with insertion of the oxo group into a C-H bond of all substrates tested (rather than olefin epoxidation for cyclohexene). A mechanism involving intermediacy of a high valent Ru(V)-oxo species is proposed for the catalytic oxidation processes.     

Platelet storage under in vitro condition is associated with calcium-dependent apoptosis-like lesions and novel reorganization in platelet cytoskeleton

Platelets are cleared from circulation after a life span of 8-10 days. The molecular mechanisms underlying platelet senescence remain poorly characterized. Here we report that, progressive functional impairment in the platelets incubated in vitro in a plasma-free isotonic medium for up to 24 h at 37 degrees C is associated with release of cytochrome c from platelet mitochondria and cleavage of procaspase-9, but without evidence of caspase-3 activation. Concomitantly, there was proteolysis of survival proteins like focal adhesion kinase, Src, gelsolin, and specific cytoskeleton-associated peptides, in a manner regulated by extracellular calcium and calpain activity. Cytoskeleton played a critical role as evidenced from the association of these proteins and their degradation products, as well as procaspase-3 and the actin regulatory small GTPase, CDC42Hs, with the cytoskeleton of the stored platelets. The cytoskeletal enrichment with specific proteins was not associated with increase in the content of F-actin and was cytochalasin-resistant, thus signifying a novel mechanism of interaction of the translocating proteins with the pre-existing cytoskeleton. There was progressive exposure of phosphatidylserine on the outer leaflet of platelet membrane and specific electron microscopic changes suggestive of apoptotic lesions. Based on these observations we discuss the caspase-independent but calpain-mediated signaling events in the stored platelets resembling the features of apoptosis in the nucleated cells.